| Summary: | Chitinases, which are crucial enzymes required for chitin degradation and reconstruction, are often selectively considered to be effective molecular targets for pest control due to their critical roles in insect development. Although the <i>Hyphantria cunea</i> chitinase gene has been reported previously, its sequence characteristics, gene function, and feasibility as a potential target for pest management were absent. In the present study, we characterized the <i>H. cunea</i> chitinase gene and designated it <i>HcCht5</i>. Phylogenic and domain structure analysis suggested that <i>HcCht5</i> contained the typical chitinase features and was clustered into chitinase group I. Tissue-specific and developmental expression pattern analysis with Real-Time Quantitative PCR (RT-qPCR) showed that <i>HcCht5</i> was mainly expressed in the integument tissues and that the transcript levels peaked during molting. RNA interference (RNAi)-mediated silencing of <i>HcCht5</i> caused 33.3% (2 ug) and 66.7% (4 ug) mortality rates after double-stranded RNA (dsRNA) injection. Importantly, the interference efficiency of <i>HcCht5</i> depended on the injection time of double-stranded RNA (dsRNA), as the pre-molting treatment achieved molt arrest more effectively. In addition, transcriptome sequencing (RNA-seq) analysis of RNAi samples demonstrated silencing of the down-regulated <i>HcCht5</i> genes related to chitin metabolism and molting hormone signaling, as well as genes related to detoxification metabolism. Our results indicate the essential role of <i>HcCht5</i> in <i>H. cunea</i> development and detail the involvement of its gene function in the larval molting process.
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